Yersinia pestis is designated by the Centers for Disease Control (CDC) and NIAID as a Category A bacterial pathogen. Y. pestis is the etiological agent of the plague (Black Death), a transmissible disease that has been responsible for millions of deaths throughout the course of history. Although the natural occurrence of this disease is now relatively rare, the possibility of terrorist groups using Y. pestis as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. The long-term goal and commercial application of this proposal is to develop a plague diagnostic detection kit. The Phase I research successfully completed the proof of principle study and generated a recombinant luxAB ('light')-tagged reporter phage for the detection of Y. pestis. Y. pestis specific phage are currently used by the CDC and the World Health Organization (WHO) as a diagnostic standard for the confirmed identification of Y. pestis; however, the phage lysis assays are laboratory based and require 2 to 3 days to complete. In contrast, the technology described in this proposal does not require sample processing, a laboratory environment, or extensive incubation periods. The Phase I results demonstrated that the luxAB reporter phage rapidly conferred a bioluminescent signal response to an attenuated Y. pestis strain and displayed potential as a plague diagnostic. The Phase II research will build upon the Phase I foundation with the goal of generating a plague diagnostic kit. The Phase II research will: (i) increase the specificity and sensitivity of the reporter phage for Y. pestis (Aim 1); (ii) demonstrate the utility of the reporter phage against a library of wild-type Y. pestis strains (Aim 2); (iii) analyze the specificity of the reporter phage for Y. pestis using a panel of pathogenic non-pestis Yersinia species (Aim 3), and (iv) analyze and optimize the ability of the detection system to function directly with clinical specimens (Aim 4). Collectively, this research will provide the foundation for a plague diagnostic kit using novel 'light producing' reporter phage that rapidly, sensitively, and specifically confer a bioluminescent phenotype to Y. pestis. The vision of the product is that it can be used as a diagnostic for suspected plague-infected patients with cultivated isolates or directly with infected clinical specimens in a laboratory setting or as a portable camera-based assay. The long-term goal of our research is to develop a reporter phage-based multiplex assay for the identification of different bacterial priority A pathogens. PUBLIC HEALTH RELEVANCE: This proposal is significant because it will generate a rapid plague diagnostic test kit. A rapid diagnostic test kit is essential in order to specifically diagnose the disease, prevent the spread of the disease, and to save lives.